Interactive Fly, Drosophila

NO and behavior

A role for the NO-cGMP pathway in mediating chemosensory activation in snail feeding is suggested by intense NADPH diaphorase staining observed in nerve fibers that project from sensory cells in the lips to the CNS and by the presence in the CNS of a NO-activated guanylyl cyclase. In in vitro preparations reduced to isolated lips and CNS, intracellular recordings were made from motoneurons driven by the interneurons of the central pattern generator (CPG) for feeding. Fictive feeding in such preparations can be recorded from these motoneurons following the application of sucrose to the lips. Sucrose activation of fictive feeding is inhibited by the NO scavenger hemoglobin, a NO synthase inhibitor and by methylene blue, an inhibitor of guanylyl cyclase. Fictive feeding in isolated lip-CNS preparations can be activated without sucrose by superfusion of NO donor molecules and by a nonhydrolyzable analog of cGMP. The feeding CPG can also be activated centrally by depolarizing a modulatory interneuron, the slow oscillator (SO). When the CPG is activated in this way, fictive feeding is not susceptible to inhibition by hemoglobin, the most potent of the inhibitors of sucrose-activated fictive feeding. Behavioral experiments on intact snails confirm the findings from in vitro experiments and show that hemoglobin prevents feeding and methylene blue significantly delays the onset of feeding. These results indicate (1) that NO is a putative chemosensory transmitter in the snail L. stagnalis, (2) that the NO-cGMP pathway can mediate chemosensory activation of specific patterns of centrally generated behavior, (3) that NO is not involved in transmission within the central network of neurons responsible for the behavior, and more generally (4) that a freely diffusing and highly reactive gaseous signalling molecule can have restricted and specific behavioral functions (Elphick, 1995).

Circadian rhythms of mammals are timed by an endogenous clock with a period of about 24 hours located in the suprachiasmatic nucleus (SCN) of the hypothalamus. Light synchronizes this clock to the external environment by daily adjustments in the phase of the circadian oscillation. The mechanism has been thought to involve the release of excitatory amino acids from retinal afferents to the SCN. Brief treatment of rat SCN in vitro with glutamate (Glu), N-methyl-D-aspartate (NMDA), or nitric oxide (NO) generators produce lightlike phase shifts of circadian rhythms. The SCN exhibits calcium-dependent nitric oxide synthase (NOS) activity. Antagonists of NMDA or NOS pathways block Glu effects in vitro, and intracerebroventricular injection of a NOS inhibitor in vivo block the light-induced resetting of behavioral rhythms. Together, these data indicate that Glu release, NMDA receptor activation, NOS stimulation, and NO production link light activation of the retina to cellular changes within the SCN, mediating the phase resetting of the biological clock (Ding, 1994).

NO and LTP and memory

Temporal correlation between pre- and postsynaptic activities is an important mechanism that regulates synaptic connectivity during development and synaptic plasticity in the adult. In developing neuromuscular junctions, postsynaptic activity is critical in functional suppression and, ultimately, elimination of the synapses. Although repetitive postsynaptic firing asynchronous to the presynaptic activity results in a persistent synaptic suppression, the underlying molecular mechanism remains unknown. Evidence that nitric oxide (NO), a free radical implicated in several forms of synaptic plasticity, may serve as a retrograde signal for activity-dependent suppression in the neuromuscular synapse. NO donors and activators of the cyclic GMP pathway suppress spontaneous and evoked synaptic currents. Moreover, the synaptic suppression induced by repetitive postsynaptic depolarization is prevented by the NO-binding protein hemoglobin and by inhibitors of NO synthase. Thus, synaptic suppression may be triggered by NO released from a postsynaptic myocyte that fires asynchronously to the presynaptic terminal (Wang, 1995).

Nitric oxide (NO) has been proposed to act as a retrograde messenger during long-term potentiation (LTP) in the CA1 region of hippocampus, but the inaccessibility of the presynaptic terminal has prevented a definitive test of this hypothesis. Because both sides of the synapse are accessible in cultured hippocampal neurons, this preparation was used to investigate the role of NO. LTP was examined following intra- or extracellular application of a NO scavanger, an inhibitor of NO synthase, and a membrane-impermeant NO donor that releases NO only upon photolysis with UV light. NO is produced in the postsynaptic neuron, travels through the extracellular space, and acts directly in the presyaptic neuron to produce long-term potentiation, supporting the hypothesis that NO acts as a retrograde messenger during LTP (Arancio, 1996).

Sheep learn to recognize the odors of their lambs within two hours of giving birth; this learning involves synaptic changes within the olfactory bulb. Specifically, mitral cells become increasingly responsive to the learned odor, which stimulates release of both glutamate and GABA (gamma-aminobutyric acid) neurotransmitters from the reciprocal synapses between the excitatory mitral cells and inhibitory granule cells. Nitric oxide (NO) has been implicated in synaptic plasticity in other regions of the brain as a result of its modulation of cyclic GMP levels. NO is involved in olfactory learning. Neuronal enzyme nitric oxide synthase (nNOS) is expressed in both mitral and granule cells, whereas the guanylyl cyclase subunits that are required for NO stimulation of cGMP formation are expressed only in mitral cells. Immediately after birth, glutamate levels rise, inducing formation of NO and cGMP, which potentiate glutamate release at the mitral-to-granule cell synapses. Inhibition of nNOS or guanylyl cyclase activity prevents both the potentiation of glutamate release and formation of the olfactory memory. The effects of nNOS inhibition can be reversed by infusion of NO into the olfactory bulb. Once memory has formed, however, inhibition of nNOS or guanylyl cyclase activity cannot impair either its recall or the neurochemical release evoked by the learned lamb odor. Nitric oxide therefore seems to act as a retrograde and/or intracellular messenger, being released from both mitral and granule cells to potentiate glutamate release from mitral cells by modulating cGMP concentrations. It is proposed that the resulting changes in the functional circuitry of the olfactory bulb underlie the formation of olfactory memories (Kendrick, 1997).

High-frequency stimulation (HFS) of corticostriatal glutamatergic fibers induces long-term depression (LTD) of excitatory synaptic potentials recorded from striatal spiny neurons. This form of LTD can be mimicked by zaprinast, a selective inhibitor of cGMP phosphodiesterases (PDEs). Biochemical analysis shows that most of the striatal cGMP PDE activity is calmodulin-dependent and inhibited by zaprinast. The zaprinast-induced LTD occludes further depression by tetanic stimulation and vice versa. Both forms of synaptic plasticity are blocked by a selective inhibitor of soluble guanylyl cyclase, indicating that an increased cGMP production in the spiny neuron is a key step. Accordingly, intracellular cGMP, activating protein kinase G (PKG), also induces LTD. Nitric oxide synthase (NOS) inhibitors block LTD induced by either HFS or zaprinast, but not that induced by cGMP. LTD is also induced by the NO donors S-nitroso-N-acetylpenicillamine (SNAP) and hydroxylamine. SNAP-induced LTD occludes further depression by HFS or zaprinast. Intracellular application of PKG inhibitors blocks LTD induced by HFS, zaprinast, and SNAP. Electron microscopy immunocytochemistry shows the presence of NOS-positive terminals of striatal interneurons forming synaptic contacts with dendrites of spiny neurons. These findings represent the first demonstration that the NO/cGMP pathway exerts a feed-forward control on the corticostriatal synaptic plasticity (Calabresi, 1999).

Postsynaptic injection of Ca(2+)/calmodulin [Ca(2+)/CaM] into hippocampal CA1 pyramidal neurons induces synaptic potentiation, which can occlude tetanus-induced potentiation. Because Ca(2+)/CaM activates the major forms of nitric oxide synthase (NOS) to produce nitric oxide (NO), NO may play a role during Ca(2+)/CaM-induced potentiation. Extracellular application of the NOS inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME) or postsynaptic co-injection of L-NAME with Ca(2+)/CaM blocks Ca(2+)/CaM-induced synaptic potentiation. Thus, NO is necessary for Ca(2+)/CaM-induced synaptic potentiation. In contrast, extracellular perfusion of membrane-impermeable NO scavengers N-methyl-D-glucamine dithiocarbamate/ferrous sulfate mixture (MGD-Fe) or 2-(4-carboxyphenyl)-4,4,5, 5-tetramethylimidazoline-1-oxyl-3-oxide (carboxy-PTIO) does not attenuate Ca(2+)/CaM-induced synaptic potentiation, even though MGD-Fe or carboxy-PTIO blocks tetanus-induced synaptic potentiation. This result indicates that NO is not a retrograde messenger in Ca(2+)/CaM-induced synaptic potentiation. However, postsynaptic co-injection of carboxy-PTIO with Ca(2+)/CaM blocked Ca(2+)/CaM-induces potentiation. Postsynaptic injection of carboxy-PTIO alone blocks tetanus-induced synaptic potentiation without affecting basal synaptic transmission. These results suggest that NO works as a postsynaptic (intracellular) messenger during Ca(2+)/CaM-induced synaptic potentiation (Ko, 1999).

Long-term potentiation, a persistent increase in synaptic efficacy, may require a retrograde signal originating in the postsynaptic cell that induces an increase in presynaptic neurotransmitter release. A mouse homozygous for a targeted null mutation in the endothelial isoform of nitric oxide synthase has been constructed. Long-term potentiation in the CA1 region of these mice is entirely absent under weak stimulation conditions. Application of a membrane-permeant guanosine-3',5'-cyclic monophosphate analog during tetanus fails to compensate for this deficit, suggesting that nitric oxide produced by endothelial nitric oxide synthase may affect long-term potentiation through a cascade that does not include guanylyl cyclase. Strong tetanic stimulation can induce robust long-term potentiation in these mice. This potentiation is not blocked by pharmacological inhibitors of nitric oxide synthase. Furthermore, mice lacking endothelial nitric oxide synthase show no shift in the frequency-response curve for the induction of long-term potentiation. Basal synaptic transmission, paired-pulse facilitation and the electrical properties of CA1 cells in these mice are similar to controls. These results support a selective role for endothelial nitric oxide synthase in long-term potentiation, but also demonstrate that nitric oxide synthase is not always involved in this process under all conditions (Wilson, 1999).

Long-term potentiation (LTP) is a potential cellular mechanism for learning and memory. The retrograde messenger nitric oxide (NO) is thought to induce LTP in the CA1 region of the hippocampus via activation of soluble guanylyl cyclase (sGC) and, ultimately, cGMP-dependent protein kinase (cGK). Two genes code for the isozymes cGKI and cGKII in vertebrates. The functional role of cGKs in LTP was analyzed using mice lacking the gene(s) for cGKI, cGKII, or both. LTP is not altered in the mutant mice lineages. However, LTP is reduced by inhibition of NO synthase and NMDA receptor antagonists, respectively. The reduced LTP was not recovered by the cGK-activator 8-(4 chlorophenylthio)-cGMP. Moreover, LTP was not affected by a sGC inhibitor. In contrast, it is effectively suppressed by nicotinamide, a blocker of the ADP-ribosyltransferase. These results show that cGKs are not involved in LTP in mice and that NO induces LTP through an alternative cGMP-independent pathway, possibly ADP-ribosylation (Kleppisch, 1999).

Pharmacological studies support the idea that nitric oxide (NO) serves as a retrograde messenger during long-term potentiation (LTP) in area CA1 of the hippocampus. Mice with a defective form of the gene for neuronal NO synthase (nNOS), however, exhibit normal LTP. The myristoyl protein endothelial NOS (eNOS) is present in the dendrites of CA1 neurons. Recombinant adenovirus vectors containing either a truncated eNOS (a putative dominant negative) or an eNOS fused to a transmembrane protein were used to demonstrate that membrane-targeted eNOS is required for LTP. The membrane localization of eNOS may optimally position the enzyme both to respond to Ca2+ influx and to release NO into the extracellular space during LTP induction (Kantor, 1996).

Nitric oxide (NO) has been implicated in hippocampal long-term potentiation (LTP), but LTP is normal in mice with a targeted mutation in the neuronal form of NO synthase (nNOS-). LTP is also normal in mice with a targeted mutation in endothelial NOS (eNOS-), but LTP in stratum radiatum of CA1 is significantly reduced in doubly mutant mice (nNOS-/eNOS-). By contrast, LTP in stratum oriens is normal in the doubly mutant mice. These results provide the first genetic evidence that NOS is involved in LTP in stratum radiatum and suggest that the neuronal and endothelial forms can compensate for each other in mice with a single mutation. They further suggest that there is also a NOS-independent component of LTP in stratum radiatum and that LTP in stratum oriens is largely NOS independent (Son, 1996).

Body size and behavioral state of C. elegans and cGMP-dependent protein kinase

The growth and behavior of higher organisms depend on the accurate perception and integration of sensory stimuli by the nervous system. Defects in sensory perception in C. elegans result in abnormalities in the growth of the animal and in the expression of alternative behavioral states. This analysis suggests that sensory neurons modulate neural or neuroendocrine functions, regulating both bodily growth and behavioral state. Genes likely to be required for these functions downstream of sensory inputs have been identified. One of these genes has been characterized as egl-4; it encodes a cGMP-dependent protein kinase. This cGMP-dependent kinase functions in neurons of C. elegans to regulate multiple developmental and behavioral processes including the orchestrated growth of the animal and the expression of particular behavioral states (Fujiwara, 2002).

Despite having a simple nervous system of 302 neurons, C. elegans is capable of perceiving and responding to a wide variety of environmental stimuli such as odorants, mechanical stimuli, food, osmotic, and ionic changes and pheromones. In C. elegans, environmental cues are detected through specialized sensory neurons. Sixty of the 302 neurons in C. elegans are ciliated sensory neurons, which are thought to be responsible for most sensory perceptions. Like many sensory neurons in other animals, the sensory cilia of these neurons are specialized structures where environmental cues, including odorants and pheromones, interact with receptor proteins (Fujiwara, 2002).

A class of mutants including che-2, osm-6, and che-3 lack a normal sensory cilium structure. The structural defects in these mutants are readily assessed because several ciliated sensory neurons take up vital dyes through the cilia, and these mutants fail to do so. As expected, mutants lacking cilia show diminished sensory responses to soluble and volatile chemicals. They have diminished responses to dauer pheromone, which induces a transition to an alternative nondeveloping dauer larva stage. It has also been reported that the cilium-defective mutants exhibit a longer life span than wild-type animals (Fujiwara, 2002).

Mutants with defects in cilium structure also exhibit abnormalities in the regulation of growth to a normal body size and in the expression of alternative states of locomotory behavior. The changes in body size in the cilium-defective mutants are not due to an inability to locate food. The results suggest that sensory perception can regulate neuroendocrine functions that determine the growth and ultimate body size of an organism. Locomotory behavior of C. elegans in the presence of food is characterized by alternating behavioral states. In one state, the animal traverses widely separated regions of the plate (roaming), and in the other state, the animal restricts its activity to a confined region (dwelling). Analysis of cilium-defective mutants reveals that defects in sensory perception result in a relative decrease in the time spent roaming. Hence, the relative time spent roaming versus dwelling may be regulated by sensory perception (Fujiwara, 2002).

A genetic analysis has been persued to determine how such changes in development and behavior are regulated by sensory perception. To identify neuronal mechanisms acting downstream of sensory perception in the regulation of these processes, a screen was performed for suppressor mutations of the che-2 small body size phenotype (chb). A subset of these suppressors also suppress the defect in locomotory behavior of che-2. One of these suppressors, chb-1 (which is allelic to egl-4), encodes a cGMP-dependent kinase. A homologous cGMP-dependent protein kinase is expressed in vertebrate brain, although the physiological functions of this kinase in the nervous system have been controversial. These results suggest that cGMP-dependent kinase is required for the processing of sensory information that is essential to multiple behavioral and developmental circuits in C. elegans (Fujiwara, 2002).

In Drosophila, strains having less cGMP-dependent protein kinase activity move less during foraging (Osborne, 1997. Sokolowski, 1998). Although the same kinase influences the movement of both species, the mechanisms may be different. In Drosophila, the general locomotory activity level during foraging is different in distinct strains. In C. elegans, transitions between alternative active and inactive states are affected in a single strain. The effects in Drosophila are also opposite in character; a decrease in this protein results in increased roaming in C. elegans but less movement in Drosophila. The expression of a cGMP-dependent protein kinase in Drosophila is observed in neuronal and nonneuronal tissues. It is not yet known where expression is required for the regulation of foraging behavior of Drosophila. It is not yet known (Osborne, 2001) where expression is required for the regulation of foraging behavior of Drosophila (Fujiwara, 2002 and references therein).

The data presented in this study suggest that EGL-4 acts in the nervous system and particularly in sensory neurons in C. elegans. This represents a novel demonstration of the role of a cGMP-dependent protein kinase in sensory neurons for modulation of sensory information. egl-4 mutant phenotypes including enhanced dauer formation at high temperature, large body size, egg-laying defects, and even chemotaxis defects are suppressed by daf-3 mutations, suggesting that daf-3 functions downstream of egl-4. daf-3 encodes a member of the SMAD protein family acting downstream of the daf-7 TGF-ß cascade and is expressed in many neurons, the intestine, the pharynx, and hypodermi. Additional genetic interactions are observed with dbl-1 and lon-1, suggesting that egl-4 may act upstream of the dbl-1 TGF-ß cascade, which is a separate cascade from the daf-7 TGF-ß cascade. Although it is unclear whether egl-4 directly regulates dbl-1 and how daf-3 may interact with the dbl-1 TGF-ß cascade, the results suggest that the EGL-4 cGMP-dependent kinase may link sensory perception to the dbl-1TGF-ß cascade (Fujiwara, 2002).

A model is proposed in which sensory perception, acting through modulation of a cGMP-dependent kinase, regulates the growth and locomotory behavior of the animal. Normally, the EGL-4 cGMP-dependent kinase functions to reduce body size and decrease roaming. In wild-type animals, EGL-4 activity would be inhibited by sensory inputs. In che-2 mutants, EGL-4 would be inappropriately activated, resulting in a small body and decreased roaming. In egl-4;che-2 double mutants, EGL-4 function is eliminated, and body size and roaming are increased. Such a model of inhibitory sensory inputs is reminiscent of phototransduction in the vertebrate retina where light sensation causes a decrease in cGMP through activation of PDE. By analyzing other chb suppressors, it should be possible to identify additional components acting downstream of sensory inputs to regulate the growth and behavioral state of C. elegans (Fujiwara, 2002).

Table of contents

Nitric oxide synthase: Biological Overview | Regulation | Developmental Biology | References

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